Regulatory T cells (Tregs) contribute to the maintenance of self-tolerance and mitigate graft-versus-host disease (GvHD), a major complication of allogeneic hematopoietic stem cell transplantation (HSCT), while preserving the beneficial graft-versus-leukemia (GvL) effect. However, in vitro expansion of the rare Treg cell subset is inefficient, costly, and time-consuming. The locus of Foxp3, the master regulator of Tregs, is unmethylated and expressed only in Tregs. We have recently reported that the hypomethylating agent azacitidine (AzaC) induces Foxp3 expression and increases Tregs in vivo, thereby mitigating GvHD without abrogating GvL in a murine allogeneic transplant model. We have also developed an in vivo imaging technique, [18FJ-FHBG-PET, to track genetically-modified T cells carrying a chimeric suicide gene (CD34- TK75). In this renewal, we will further define the optimal conditions for AzaC-induced immune suppression in murine allogeneic transplant models (Aimi), confirm that similar effects can be demonstrated in human T cells using informative xenograft GvHD/leukemia models developed in our lab (Aim 2), and validate the effects of AzaC in a pilot clinical trial (Aim 3). In the clinical trial proposed in Aim 3, we propose to give patients with relapsed AML or MDS after HSCT a donor lymphocyte infusion containing T cells that are transduced with our CD34-TK75 suicide/imaging gene; half will be treated with AzaC. Our hypothesis is that AzaC will convert the T cells into FoxP3+ Tregs that will control the alloreactive T cells thus mitigating GvHD without abrogating GvL effects. The ability of non-invasive [18F]-FHBG-PET imaging to measure the reconstitution, expansion and persistence of adoptively transferred CD34-TK75+ tracer T cells in patients may have significant clinical utility for providing early predictions of GvHD and AzaC treatment efficacy. RELEVANCE (See instructions): If successful, the observation of a GvHD profile by [18FJ-FHBG-PET imaging would herald the onset of severe GvHD and the need for initiation of GvHD prophylaxis. In contrast, observation of a No GvHD profile would predict negligible to limited GvHD and permit the continued observation of recipients without immunosuppressive medications, thereby permitting a more sustained GvL effect.